The existence of neurotransmitter receptor heteromers is becoming broadly accepted and their functional significance is being revealed. We now define receptor heteromers as a macromolecular complex composed of at least two (functional) receptor units with biochemical properties that are demonstrably different from those of its individual components (1). The occurrence of receptor heteromers with different pharmacological and signaling properties opens a complete new field to search for novel drug targets useful against a variety of neuropsychiatric disorders with potentially less side effects (1,2). We have previously hypothesized that neurotransmitter heteromers play a key integrative role in local modules, which we have defined as a minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit. Our recent work is related to the identification and study of the neurotransmitter receptor heteromers that modulate the function of the striatal spine modules (3). We believe that understanding the integrated function of the striatal spine modules will have important implications for the study of basal ganglia function and disfunction, including drug abuse. The main neurotransmitters influencing the striatal spine modules are dopamine, glutamate, adenosine, GABA, acetylcholine, endocannabinoids, serotonin and histamine. In previous studies we discovered the existence of the following receptor heteromers localized in different elements of the striatal spine module: adenosine A2A-dopamine D2, adenosine A1-dopamine D1, A1-A2A, A2A-metabotropic glutamate mGlu5, D2-nicotinic acetylcholine (alpha4-beta2), A2A-cannabinoid CB1, D1-dopamine D3 and D2-histamine H3 receptor heteromers and also the A2A-D2-CB1 receptor heteromultimer (see previous annual report). During the last year we also demonstrated the existence of D1-H3 receptor heteromers (4) and D2-mGlu5 receptor heteromers and A2A-D2-mGlu5 receptor heteromultimers (5). The D1-H3 receptor heteromer provided a striking example of the completely new functions that receptor heteromers can acquire compared with those of its individual receptor units. Activation of histamine H3 receptors did not lead to signalling towards the MAPK pathway unless dopamine D1 receptors were co-expressed. Also, dopamine D1 receptors, usually coupled to Gs proteins and leading to increases in cAMP, did not couple to Gs but to Gi in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor (4). Thus, D1H3 receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a Gs-independent and Gi-dependent manner. By using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of D2 and mGlu5 receptors in living cells (5). Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique (SRET), allowed us to detect the occurrence of A2A-D2-mGlu5 receptor heteromultimers. By using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of the striatal spine module (5). Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of A2A, D2 and mGlu5 receptors in rat striatum homogenates (5), strongly suggesting the existence of A2A-D2-mGlu5 receptor heteromultimers in the native brain tissue, which can constitute important therapeutic targets for basal ganglia disorders, schizophrenia and drug addiction. The A2A-D2 receptor heteromer has been our most studied receptor heteromer. In the brain, A2A and D2 receptors are highly expressed in one type of striatal neuron, the GABAergic enkephalinergic neuron. This type of neuron constitutes almost half the neuronal population in the striatum and its malfunction plays a key role in the pathogenesis of basal ganglia disorders (such as Parkinsons disease and Huntingtons chorea) and most probably in obsessive-compulsive disorders, schizophrenia and drug addiction. We have previously found key epitopes in their intracellular domains that are involved in the heteromerization. Those are an arginine-rich epitope in the amino-terminal part of the long 3rd intracellular loop (3IL) of the D2 receptor and a phosphorilable serine in the distal part of the carboxy terminus of the A2A receptor. These epitopes establish strong electrostatic interactions. During the last year we have provided direct in situ evidence for a fundamental role of these epitopes in the function of the A2A-D2 receptor heteromer. Thus patch-clamp experiments in identified GABAergic enkephalinergic neurons demonstrated that disruption of the arginine-phosphate interaction in A2A-D2 receptor heteromers (by intracellular addition of small peptides with the same sequence than the receptor epitopes involved in the Arg-Phosphate interaction) completely eliminates the ability of the A2A receptor to antagonistically modulate the D2 receptor-mediated inhibition of neuronal excitability (6). By using BRET and SRET techniques we also studied the role of Ca2+ in the modulation of the quaternary structure of the A2A-D2 receptor heteromer, which was found to depend on the binding of calmodulin (CaM) to a proximal portion of the carboxy terminus of the A2A receptor in the A2A-D2 receptor heteromer (7). Importantly, the changes in quaternary structure correlate with changes in function. A Ca2+/CaM-dependent modulation of MAPK signaling upon agonist treatment could only be observed in cells expressing A2A-D2 receptor heteromers (7). These studies provide a first example of a Ca2+-mediated modulation of the quaternary structure and function of a receptor heteromer. We have also continued with our studies about the role of striatal A2A receptors in experiments in collaboration with the Department of Neurology of Johns Hopkins University School of Medicine and with the Faculty of Medicine at the University of Lisbon. We have found that an iron-deficient diet associated with low levels of iron in the brain produces a pronounced increase in the density of A2A receptors (8). Experiments in mammalian cells demonstrated that this is a cellular phenomenon, since treatment with iron chelators in cells constitutively expressing A2A receptors produce the same effect (8). These results could have implications for the treatment of iron-linked neurological disorders, such as Restless Legs Syndrome. Finally, we found that striatal presynaptic A2A receptors, which we have previously demonstrated that controls glutamate release, play a necessary permissive role in the ability of glial cell line-derived neurotrophic factor (GDNF) to modulate cortico-striatal glutamatergic neurotransmission (9).